The present invention is concerned with angiogenesis broadly and with tumor angiogenesis directly; and is focused on means and methods for inhibiting tumor angiogenesis involving vascular endothelial growth factor (xe2x80x9cVEGFxe2x80x9d) and integrin heterodimer surface receptors found in the vasculature of a living subject.
Angiogenesis, the formation of new capillaries and blood vessels, is a complex process first recognized in studies of wound healing and then with investigations of experimental tumors. Angiogenesis involves extracellular matrix remodeling, endothelial cell migration and proliferation, and functional maturation of endothelial cells into mature blood vessels [Brier, G. and K. Alitalo, Trends Cell Biol. 6: 454-456 (1996)]. Although the process generally has been studied for more than 50 years, the existence and in-vivo effects of several discrete angiogenic factors have been identified just over a decade ago [Folkman, J. and M. Klagsburn, Science 235: 444-447 (1985)]. Clearly, in normal living subjects, the process of angiogenesis is a normal host response to injury; and as such is an integral part of the host body""s homeostatic mechanisms.
In distinction, tumor angiogenesis is the specific development in-vivo of an adequate blood supply for a solid tumor mass; and the growth of a tumor in-vivo beyond the size of a few millimeters in diameter is believed to be dependent upon the existence, maintenance, and continued development of sufficient and functional blood vasculature in-situ. In a variety of experimental tumor systems, tumor survival and growth has been linked with new capillary and new blood vessel formation. Histological examination of such neoplasms has revealed that tumor cells typically surround blood capillaries in a cylindrical configuration with a radius not exceeding about 200 micrometersxe2x80x94the critical travel distance for diffusion of molecular oxygen [Folkman, J., Cancer Res. 46: 467-473 (1986)]. Moreover, in the cancer patient, tumor angiogenesis originates at least in part from the sprouting of new capillaries and blood vessels directly from the pre-existing and functional normal vasculature; and possibly also from stem cells existing in the blood. Tumor angiogenesis thus involves endothelial cell penetration of the vascular basement membrane in a preexisting blood vessel; followed by endothelial cell proliferation; and then by an invasion of the extracellular matrix surrounding the blood vessel to form a newly created vascular spout [Vernon, R. and E. H. Sage, Am. J. Pathol. 147: 873-883 (1995); Auspunk, D. H. and J. Folkman, Microvasc. Res. 14: 53-65 (1977)].
A number of different biologically active and physiologically functional molecular entities appear to be individual factors of angiogenesis. Among these are the biologically active classes of substances known as vascular endothelial growth factor and the integrin protein family of cell surface receptors. Each of these two classes will be summarily reviewed as to their conventionally known properties and functions.
Vascular Endothelial Growth Factor
Vascular endothelial growth factor (hereinafter xe2x80x9cVEGFxe2x80x9d), also known as vascular permeability factor, is a 34-45 kilodalton dimeric glycoprotein; is a cytokine; and is a potent inducer of microvascular hyperpermeability. As such, VEGF is believed to be responsible for the vascular hyperpermeability and consequent plasma protein-rich fluid accumulation that occurs in-vivo with solid tumors and ascites tumors [Senger et al., Science 219: 983-985 (1983); Dvorak et al., J. Immunol. 122: 166 (1979); Nagy et al., Biochem. Biophys. Acta. 948: 305 (1988); Senger et al., Federation Proceedings 46: 2102 (1987)]. On a molar basis, VEGF increases microvascular permeability with a potency which is typically 50,000 times that of histamine [Senger et al., Cancer Res. 50: 1774-1778 (1990].
Vascular endothelial growth factor is also noted for its mitogenic effects on vascular endothelial cells (hereinafter xe2x80x9cECxe2x80x9d). VEGF is a specific EC mitogen which stimulates endothelial cell growth and promotes angiogenesis in-vivo [Conn et al., Proc. Natl. Acad. Sci. USA 87: 2628-2632 (1990); Ferrara et al., Biochem. Biophys. Res. Comm. 161: 851-858 (1989); Gospodarowicz et al., Proc. Natl. Acad. Sci. USA. 86: 7311-7315 (1989); Keck et al., Science 264: 1309 (1989); Leung et al., Science 246: 1306 (1989); Connolly et al., J. Clin. Invest. 84: 1407-1478 (1989)]. In addition, VEGF exerts a number of other effects on endothelial cells in-vitro. These include: an increase in intracellular calcium; a stimulation of inositol triphosphate formation; a provocation of von Willebrand factor release; and a stimulation of tissue factor expression [Brock et al., Am. J. Pathol. 138: 213 (1991); Clauss et al., J. Exp. Med. 172: 1535 (1990)].
Vascular endothelial growth factor elicits potent angiogenic effects by stimulating endothelial cells through two receptor tyrosine kinases, Flt-1 and KDR/Flk-1 [Dvorak et al., Am. J. Pathol. 146: 1029-1039 (1995); Mustonen, T. and K. Alitalo, J. Cell Biol. 129: 895-898 (1996)]. Although there are potentially numerous angiogenesis factors, considerable evidence has accumulated indicating that VEGF is a cytokine of importance both for neovascularization in the medically normal adult and for development of embryonic vasculature. VEGF angiogenic activity has been demonstrated in several experimental models including the chick chorioallantoic membrane [Whiting et al., Anat. Embryol. 186: 251-257 (1992)]; rabbit ischemic hind limb [Takeshita et al., J. Clin. Invest. 93: 662-670 (1994)]; tumor xenografts in mice [Potgens et al., Biol: Chem. Hoppe. Seyler 376: 57-70 (1995); Claffey et al., Cancer Res. 56: 172-181 (1996)]; and a primate model of iris neovascularization [Tolentino et al., Arch. Ophthalmol. 114: 964-978 (1996)]. Additionally, both infusion of exogenous VEGF and overexpression of VEGF endogenously were found to induce hypervascularization of avian embryos [Drake et al., Proc. Natl. Acad. Sci. USA 92: 7657-7661 (1995); Flamme et al., Dev. Biol. 171: 399-414 (1995)].
Evidence supporting the importance of VEGF for angiogenesis generally also has come from analyses of VEGF and VEGF receptor expression. These investigations have established that elevated expression of VEGF and its receptors correlate both temporally and spatially with vascularization during embryogenesis [Millauer et al., Cell 72: 835-846 (1993); Peters et al., Proc. Natl. Acad. Sci. USA 90: 8915-8919 (1993)]; and also with the angiogenesis associated with wound healing [Brown et al., J. Exp. Med. 176: 1375-1379 (1992)]; cancer [Brown et al., Cancer Res. 53: 4727-4735 (1993)]; rheumatoid arthritis [Fava et al., J. Exp. Med. 180: 341-346 (1994)]; psoriasis [Detmar et al., J. Exp. Med. 180: 1142-1146(1994)]; delayed-type hypersensitivity reactions [Brown et al., J. Immunol. 154: 2801-2807 (1995)]; and proliferative retinopathies [Aiello et al., N. Eng. J. Med. 331: 1480-1487 (1994); Pierce et al., Proc. Natl. Acad. Sci. USA 92: 905-909 (1995)]. Thus, VEGF appears not only to promote angiogenesis in a variety of experimental systems, but also appears to be overexpressed in a diversity of settings in which neovascularization is prominent.
VEGF is typically synthesized and secreted in-vivo by a variety of cultured tumor cells, transplantable animal tumors, and many different primary and metastatic human tumors [Dvorak et al., J. Exp. Med. 174: 1275-1278 (1991); Senger et al., Cancer Res. 46: 5629-532 (1986); Plate et al., Nature 359: 845-848 (1992); Brown et al., Am. J. Pathol. 143: 1255-1262 (1993)]. Solid tumors, however, must generate a vascular stroma in order to grow beyond a minimal size [Folkman, J. and Y. Shing, J. Biol. Chem. 267: 10931-10934 (1992)].
VEGF today is believed able to be a central mediator of angiogenesis generally as well as of tumor angiogenesis in particular. Monoclonal antibody directed against VEGF has been shown to suppress growth and decrease the density of blood vessels in experimental tumors [Kim et al., Nature 362: 841-844 (1993)].
It will be noted and appreciated also that many research investigations reported in the scientific and patent literature have employed antibodies raised against VEGF in order to identify and characterize the functions, properties, and attributes of the VEGF molecule in-vivo. Merely illustrating the range and variety of these investigations and published reports are the following: Preparation of specific antibodies [U.S. Pat. No. 5,036,003]; use of monoclonal antibodies to suppress growth and decrease density of blood vessels in tumors [Kim et al., Nature 362: 841-844 (1 993)]; inhibition of tumor growth and metastasis by antibody to VEGF [Asano et al., Cancer Res. 55: 5296-5301 (1995)]; inhibition of VEGF activity with specific antibodies [Sioussat et al., Arch. Biochem. Biophys. 301: 15-20 (1993)]; the structure of solid tumors and their vasculature [Dvorak et al., Cancer Cells 3: 77-85 (1993)]; and the distribution of VEGF in tumors and the concentration of VEGF in tumor blood vessels [Dvorak et al., J. Exp. Med. 174: 1275-1278 (1991)]. The text of each and all of these cited publications concerning VEGF is expressly incorporated by reference herein.
The Integrin Protein Family
Integrins are a specific family of cell surface receptors which function in-vivo as adhesive molecules for a large variety of different compounds and ligands. As a member of this specific receptor family, each integrin entity chemically is a heterodimeric glycoprotein; and is structurally composed of two different non-covalently linked protein subunits, each of the individual subunit moieties being chosen from among the alternative members forming a discrete 130-210 kilodalton xe2x80x9calphaxe2x80x9d (xcex1) subunit group and the individual members forming another distinct 95-130 kilodalton xe2x80x9cbetaxe2x80x9d (xcex2) subunit group. The overall structure of an integrin receptor molecule generally is illustrated by FIG. A [reproduced from Hynes, R. O., Cell 48: 549-554 (1987); see also Springer, T. A., Fed. Proc. 44: 2660-2055 (1985); Hynes, R. O., Cell 69: 11-25 (1992); Ruoslahti et al., Kidney Internatl. 45: S17-S22 (1994); and INTEGRINS: Molecular and Biological Responses to the Extracellular Matrix, (Cheresh and Mecham, editors), Academic Press, 1994.
As seen in FIG. A, the alpha and beta subunits are joined in a non-covalent linkage to form a unitary wholexe2x80x94i.e., the heterodimer. Each subunit has a transmembrane segment (shown in FIG. A as a dark area); a small C-terminal cytoplasmic domain (shown in FIG. A as a stippled area); and a large N-terminal extracellular domain. The beta (xcex2) subunits as a group typically contain sequences of extensive intrachain disulphide bonding, including four repeated regions of a forty amino acid cysteine-rich segment (shown in FIG. A as a crosshatched area). Also, some alpha (xcex1) subunit members of the group are cleaved posttranslationally to provide a heavy chain and a light chain linked by internal disulphide bonding to form the complete subunit entity. For a more detailed description of the integrin molecular structure, see Hynes, R. O., Cell 48: 549-554 (1987) and the references cited therein; Hynes, R. O., Cell 69: 11-25 (1992); Ruoslahti et al., Kidney Internatl. 45: S17-S22 (1994); and INTEGRINS: Molecular and Biological Responses to the Extracellular Matrix, (Cheresh and Mecham, editors), Academic Press, 1994.
It is essential to recognize also that each alpha subunit group and each beta subunit group has its own distinctive members, each of which can become non-covalently linked to more than one member of the corresponding subunit type. At present, the alpha subunit group comprises not less than fourteen (14) different entities; while the beta subunit group comprises not less than eight (8) different members. A representative listing and correlation of the presently recognized possible combinations and permutations of individual xcex1 and xcex2 subunits is shown by FIG. B. [reproduced in part from INTEGRINS: Molecular And Biological Responses to the Extracellular Matrix, (Cheresh and Mecham, editors), Academic Press, 1994, (preface page xii)].
The recognized biological role and in-vivo function of the integrin protein family are as cell surface receptors for cell-to-cell or cell-to-matrix interactions. Many of the individual integrin heterodimers comprising the family as a whole were first identified by their ability to bind with one specific ligand or matrix glycoprotein extracellularly. In this manner, the individual integrin heterodimers (each comprised of different xcex1 and xcex2 subunits) have demonstrated a variety of unique and alternative specific binding affinities and capacities for a diverse range of singular extracellular ligands in-vivo. The conventionally known range of such extracellular ligands presently includes: laminin, collagen, fibronectin, vitronectin, epiligin, entactin, merosin, kalinin, invasin, tenascin, osteopontin, thrombospondin, adenovirus penton base, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and von Willebrand factor. A representative listing of the individual xcex1 and xcex2 subunits composing the integrin unit with the corresponding specific bind affinity ligand is presented by FIG. C [also reproduced in part from INTEGRINS: Molecular and Biological Responses to the Extracellular Matrix, (Cheresh and Mecham, editors), Academic Press, 1994, (preface page xii)].
In addition, for the purposes of clarity and avoidance of misunderstandings or ambiguities, it is necessary to note and appreciate that the reported research investigations of what are now recognized as integrin protein molecules were pursued by different persons working in different scientific fields for a variety of different purposes. As a unfortunate consequence of this historical development of the integrin field, a series of different and alternative titles were originally given and applied to substances thought first to be differentxe2x80x94but which were subsequently found to be a single chemical structure and composition alone. This multiple naming and title designation occurrence was recognized in the literature very early; and a major effort was undertakenby 1987 to reconcile the various designations into a more consistent and uniform naming system, as is examplified by Table 1 below [reproduced from Hynes, R. O., Cell 48: 549-554 (1987)]. Thus, as Table 1 shows, the xcex11xcex21 integrin molecule was also known in 1987 as xe2x80x9chuman very late activation protein 1 complexxe2x80x9d or VLA-1. Similarly, the xcex12xcex21 integrin unit in 1987 was also alternatively titled xe2x80x9cplatelet membrane glycoprotein Ia-IIa complexxe2x80x9d or GPIa/IIa; and as xe2x80x9chuman very late activation protein 2 complexxe2x80x9d or VLA-2; and also as xe2x80x9cfibroblast extracellular matrix receptor IIxe2x80x9d, a misnomer of its true binding affinity (as shown by FIG. C).
The integrin protein family as a whole, being cell surface receptors for specific extracellular matrix ligands, has been implicated in the processes of extracellular matrix remodeling, in endothelial cell migration, and in the function maturation of new endothelial cells into mature blood vesselsxe2x80x94the complex process of angiogenesis generally. See for example, Hynes, R. O., Cell 69: 11-25 (1992); Ruoslahti et al., Kidney Internatl. 45: S17S22 (1994); and Schwartz et al., Ann. Rev. Cell Dev. Biol. 11: 549599 (1995). Also published reports of targeted gene deletion of xcex15 and xcex1v integrin subunits in living mice apparently resulted in embryonic vascular defects [Hynes, R. O., Develop. Biol. 180: 402-412 (1996)]; and an antibody which broadly inhibited members of the xcex21 subunit was shown to inhibit development of the embryonic vasculature [Drake et al., Develop. Dyn. 193: 83-91 (1992)].
In addition, other reported investigations employing a variety of different experimental models have demonstrated that an inhibition of tumor angiogenesis and of normal vasculature development can be achieved using an anti-xcex1vxcex23 blocking antibody [Brooks et al., Science 264: 569-571 (1994); Brooks et al., Cell 79: 1157-1164 (1994), Brooks et al., J. Clin. Invest. 96: 1815-1822 (1995); Drake et al., J. Cell Sci. 108: 2655-2661 (1995)]; as well as by using an ant-xcex1vxcex25 blocking antibody [Friedhandler et al., Science 270: 1500-1502 (1995)].
The xcex2 subunit grouping in particular appears to have become a favored target of current research efforts. Thus, for example, cyclic peptide compounds have been developed which can inhibit xcex21 and xcex22 mediated adhesion [PCT Int. Pub. No. WO 96/40781 dated Dec. 19, 1996]. Also, the function of the arginine-glycine-aspartic acid (RGD) amino acid sequence as a specific recognition sequence within ligands binding to xcex23 subunits has been the focus of several different recent innovations and novel peptide compounds. [PCT Int. Pub. No. WO 97/08203 dated Mar. 6, 1997; PCT Int. Pub. No. WO 97/14716 dated Apr. 24, 1997; see also U.S. Pat. Nos. 5,192,746; 5,294,713; and 5,260,277.]
To illustrate the general state of the pertinent field and to provide a greater degree of descriptive detail generally regarding conventionally known properties, capabilities and chemical composition and structure for the alpha (xcex1) subunit group and membership; the beta (xcex2) subunit group and membership; and the integrin protein family as a wholexe2x80x94the reader is directed to the following representative publications, all of which are also expressly incorporated by reference herein: Santoro, S. A., Cell 46: 913-920 (1986); Mould et al., J. Biol. Chem. 265: 4020-4024 (1989); Wagner et al., J. Cell Biol. 109: 1321-1220 (1989); Guan, J. L. and R. O. Hynes, Cell 60: 53-61 (1990); Staaz et al., J. Biol. Chem. 265: 4778-4781 (1990); Carter et al., J. Cell Biol. 110: 1387-1404 (1990); Wayner, E. A. and W. G. Carter, J. Cell Biol. 105: 1873-1884 (1987); Fitzpatrick et al., The Structure and Development of Skin, (Jeffers, Scott and White, editors), McGraw-Hill Co., 1987; Davis et al., Biochem. Biophys. Res. Comm. 182: 1025-1031 (1992); Elices, M. J. and M. E. Hemler, Proc. Natl. Acad. Sci. USA 86: 9906-9910 (1989); Languino et al, J. Cell Biol. 109: 2455-2462 (1989); Takada, Y. and M. E. Hemler, J. Cell Biol. 109: 397-407 (1989); Ignatius et al., J. Cell Biol. 111: 709-720 (1990); Kirchhofer et al., J. Biol. Chem. 265: 615-618 (1990); Kramer et al., J. Cell Biol. 111: 1233-1243 (1990); Tawil et al., Biochemistry 29: 6540-6544 (1990); Kern et al., J. Biol. Chem. 269: 22811-22816 (1994); Briesewitz et al., J. Biol. Chem. 268: 2989-2996 (1993); Sriramarao et al., J. Cell Sci. 105: 1001-1012 (1993); Gardner et al., Develop. Biol. 175: 301-313 (1996); Wong et al., Cell Adhesion Commun. 4: 201-221 (1996); and Mercurio A. M., Trends Cell Biol. 5: 419-423 (1995); and Senger et al., Am. J. Path. 149: 293-305 (1996).
In sum therefore, despite the very considerable body of presently accumulated information and knowledge regarding vascular endothelial growth factor and the integrin heterodimer family, the relationships or involvements between these two classes of biologically active substances have been explored only minimally to date. Equally important, any respective role or function in-vivo conventionally known for either VEGF or the integrin molecules individually has almost always focused on the properties and capabilities of each class of substance alone and without regard or attention to the possible influence of the other. This perspective and circumstance is true for angiogenesis broadly as well as for tumor angiogenesis in particular. For these reasons accordingly, were an effective and reliable method to be developed for an inhibition of tumor angiogenesis which utilized and depended upon a direct and dependent relationship in-vivo between VEGF and specifically induced and expressed integrin cell surface receptorsxe2x80x94such an inhibitory methodology would be recognized and appreciated as an unforeseen and uncontemplated innovation by workers in this technical field.
The present invention has multiple aspects and alternative definitions. A first aspect of the invention provides a method for inhibiting tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) and integrin cell surface receptors expressed in the vasculature of a living subject, said method comprising the steps of:
allowing mobile VEGF secreted by a tumor mass present within the body of a living subject to become bound in-vivo to the surface of endothelial cells in a tumor-associated blood vessel;
permitting said bound VEGF to induce the expression of specified integrin heterodimers on the endothelial cell surface of the tumor-associated blood vessel in-vivo, said induced and expressed integrin heterodimers being selected from the group consisting of integrins composed of xcex11 and xcex12 integrin subunits; and then
administering at least one antagonistic antibody preparation effective against said induced and expressed specified integrin heterodimers on the endothelial cell surface to the living subject such that tumor angiogenesis is inhibited in-vivo, said antagonistic preparation comprising at least one antibody specific for an integrin subunit selected from the group consisting of the xcex11 and xcex12 integrin subunits.
A second aspect of the invention provides an alternative method for inhibiting tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) and integrin cell surface receptors expressed in the vasculature of a living subject, said alternative method comprising the steps of:
allowing mobile VEGF secreted by a tumor mass present within the body of a living subject to become bound in-vivo to the surface of endothelial cells in a tumor-included blood vessel;
permitting said bound VEGF to induce the expression of specified integrin heterodimers on the endothelial cell surface of the tumor-included blood vessel in-vivo, said induced and expressed integrin heterodimers being selected from the group consisting of integrins composed of xcex11 and xcex12 integrin subunits; and then
administering at least one antagonistic antibody preparation effective against said induced and expressed specified integrin heterodimers on the endothelial cell surface to the living subject such that tumor angiogenesis is inhibited in-vivo, said antagonistic preparation comprising at least one antibody specific for an integrin subunit selected from the group consisting of the xcex11 and xcex12 integrin subunits.